First Report of Colletotrichum fructicola Causing Fruit Rot and Leaf-Tip Dieback on Pineapple in Northern Thailand

Pineapple is one of the most economically important fruits in tropical countries, particularly in Thailand. Canned pineapple is currently Thailand’s main exported commodity to many countries, including the United States, Russia, Germany, Poland, and Japan. Fungal diseases are considered a permanent threat to fruits in the pre- and post-harvest stages, leading to considerable economic losses. Fungal disease is one of the primary causes of massive yield losses in pineapples around the world. Colletotrichum species are the most common fungal pathogens affecting different tropical fruits. Although there are many reports regarding Colletotrichum species associated with pineapple, they do not have molecular data to confirm species identification. However, the occurrence of Colletotrichum species on pineapple has not been reported in Thailand so far. In this study, we isolated and identified Colletotrichum fructicola on pineapple in northern Thailand and have proven its pathogenicity to the host. This is the first report of the occurrence of Colletotrichum in pineapple, based on morpho-molecular approaches.


Introduction
Pineapple (Ananas comosus (L.) Merr.) is one of the edible and nutritious fruits of the Bromeliaceae, grown in tropical and subtropical countries [1]. Asia, South Central America, and Africa are the world's leading areas producing this fruit [2]. Pineapple is the second largest tropical crop in the world [2] and the third most consumed fruit worldwide [3]. Brazil, China, Costa Rica, India, the Philippines, and Thailand are the top pineappleproducing countries [1]. Pineapple could be eaten as a fresh fruit or selected as a basic raw ingredient used in the confectionery industry [4][5][6]. The fruit contains immense nutrients and is abundant in vitamins A, C, B1, and B6 [7][8][9]. It also has proteins, carbohydrates, fiber, copper, manganese, and several minerals [5,10].
In Thailand, pineapple products have been regarded as economic commodities for export [11]. Thailand is currently the second-largest supplier of processed pineapple fruit in the world. Production areas for pineapple in Thailand have been divided into northern, northeastern, central, and southern parts [12]. Most cultivated areas are in Prachuap Khiri Khan, Rayong, Ratchaburi, and Chonburi provinces [11]. Pineapple is classified into five groups based on the morphology of the leaf and fruit, namely Abacaxi, Cayenne, Maipure or Perolera, Queen, and Spanish [13]. Among these, Cayenne, Queen, and Spanish are cultivated in Thailand [14]. Nang Lae district in Chiang Rai province is the most pineapple-cultivating area, and Nang Lae (Cayenne group) and Phu Lae (Queen group) are the most common varieties in northern Thailand (http://www.doa.go.th/; https://www.saio.co.th/; accessed on 12 December 2022). The pineapple-growing area in Thailand is around 72,656 hectares, with an annual production of 1,680,884 metric tons. The total canned pineapples exported from Thailand were 290,524 metric tons in 2020, valued at about 345 million US dollars [15].

Morphological Studies
Following 7-14 days of incubation, morphological features including culture (color and growth rate) and microscopic features (conidiogenous cells and conidial measurements, appressoria measurements) were recorded for P76 (MFLU 22-0302) and P76-3 (MFLU 22-0303). The two strains, P76 and P76-3 were isolated from rotting pineapple fruit and leaf dieback, respectively. Morphological comparisons of P76 and P76-3 were performed on 14-day-old cultures grown on PDA at 25 • C ± 2 • C. There were minor differences in size of conidia and conidiogenous cells and also in appressoria shape which are very common in a specific species within the C. gloeosporioides species complex. Finally, these two isolates were identified as C. fructicola based on morpho-molecular evidence.

Pathogenicity Assay
Pathogenicity test results related to strain P76 showed that this strain can cause disease on both wounded and non-wounded host leaves. The wounded leaves inoculated with P76 showed dieback symptoms 4 days after the inoculation, whereas those of the non-wounded leaves showed symptoms 6 days after the inoculation. However, the symptoms continued to spread in both wounded and non-wounded leaves after 10 days. After 11 days, aerial mycelia started to grow on the surface of the symptomatic area in both wounded and non-wounded leaves. (Figure 4, P76/W, P76/NW).
Six days after the inoculation, symptoms on the injured leaves treated with P76-3 were only present at the border of the mycelial plugs ( Figure 4, P76-3/W). The non-wounded leaves inoculated with P76-3 remained asymptomatic during the test (Figure 4, P76-3/NW) along with the negative control treatments. The re-isolated fungi were identified as C. fructicola according to morphological characteristics.

Discussion
In this study, the diseased leaves showing dieback symptoms and rotting fruits of pineapple were collected in Chiang Rai province, northern Thailand. Based on a direct isolation from the fruiting bodies ( Figure 2) and an indirect isolation of infected leaves via tissue culture (Figure 3), we obtained two Colletotrichum isolates. Jayawardena et al., (2021) recommended using a polyphasic approach to identify Colletotrichum species [43]. We used both morphological examination and multi-loci molecular analysis for specieslevel identification. The phylogenetic analysis of a combined dataset of ITS, ACT, CHS-1, GAPDH, and TUB2 showed that the two isolates of Colletotrichum associated with pineapple belong to C. fructicola (Figure 1). Morphological studies also confirmed the phylogenetic results. Colletotrichum species have a wide host range and geographical distribution worldwide [16,44]. However, the occurrence of Colletotrichum species on pineapple has not so far been reported in Thailand. In the USDA host fungal database, there are 12 records of Colletotrichum species on Ananas sp. [45]. Colletotrichum ananas was reported in India [46], while C. truncatum was reported to cause leaf-tip dieback in Malaysia [45]. Colletotrichum gloeosporioides has been recorded to cause anthracnose on pineapple in China and the United States [45]. Colletotrichum gloeosporioides was also recorded from Ananas spp. in Brazil on pineapple leaves. Colletotrichum sp. was identified in Cuba, India, Korea, Panama, and the West Indies [47]. However, these species were identified based only on morphology.
In recent decades, identification based on morphology has led to the misidentification of fungal pathogens [43]. In plant pathology, correct identification of fungal species is a fundamental step that links information concerning biology, host range, distribution, pathogenicity and food security [43,48], indicating the importance of species identification to barricade future afflictions provoked by these pathogens [49]. Moreover, emerging pathogens have also been increasing threats during the last decade [24]. Colletotrichum, being a complex genus, shares overlapping morphological characteristics among species. Colletotrichum fructicola belongs to the gloeosporioides species complex, which comprises fruit rots and post-harvest pathogens. Since this species complex is the most confusing within the Colletotrichum genus, morphological identification alone cannot be trusted to identify the species correctly. It follows that the use of a polyphasic approach in plant pathology is crucial for the precise identification and naming of fungi, which will advance the management and control of both recognized and newly emerging diseases [43]. Furthermore, a polyphasic approach using molecular analysis is an effective tool to identify cryptic species and estimate species diversity [49].
Among Colletotrichum species in the gloeosporioides species complex, C. fructicola has a very broad host range, isolated from more than eight plant families as endophytes and plant pathogens [48]. It has been reported from America, Asia, Africa, Europe, and Oceania [38]. In this study, we isolated and illustrated C. fructicola as the first report of Colletotrichum species associated with pineapple in Thailand, based on molecular and morphological analyses. Furthermore, the pathogenicity tests proved that the isolates are pathogens to pineapple (Figure 4).
The results of the present study can be useful for pathologists in understanding the fungal pathogen diversity associated with pineapple, disease management and quarantine purposes. Many new Colletotrichum species have been introduced in 2022 (https://www. mycobank.org/; accessed on 8 December 2022), and there are potentially many novel species of Colletotrichum that remained undiscovered [49]. Additionally, many studies reported new hosts for existing Colletotrichum species [50][51][52][53][54][55][56][57][58], broadening their host and geographical ranges. Therefore, more investigations on the isolation and identification of Colletotrichum associated with pineapple can improve our knowledge about fungal diversity and host range and will potentially lead to the discovery of novel species of Colletotrichum.

Sample Collection, Examination and Isolation
In order to isolate the fungal pathogens associated with pineapple plants, pineapple leaves with diebacks and leaf spots, and rotting fruits were considered. In total, 10 symptomatic leaves and 12 rotting pineapple fruits were collected during June-July 2022 from organic farms in Ban Du and Nang Lae sub-districts, Mueang Chiang Rai district, Chiang Rai province, northern Thailand. The samples were kept in plastic bags labelled with the collection date, collection site and host name before being transported to the laboratory for further examination. The fruiting bodies on natural substrates were observed and photographed using a stereomicroscope (OLYMPUS SZX16; Tokyo, Japan). Morphological features were observed using a LEICA-EZ4 stereomicroscope and photographed with an optical microscope equipped with a Nikon DS-Ri2 camera. The photo plates were made by the Adobe Photoshop v.21.1.2 software, and the scales were measured by the Tarosoft (R) Image Frame Work software.
Direct isolation and indirect isolation (tissue isolation) were used to obtain cultures [59]. Further, 30 mm 2 leaf fragments were cut from the margins of lesions for tissue isolation, and were sterilized by submerging in 70% ethanol for 2 min, 10% sodium hypochlorite solution for 60 s, followed by three times rinsing in sterile distilled water for 60 s [32].

Phylogenetic Analyses
Sequences for the selected strains were obtained from GenBank (Table 3), according to blast-searching and related publications [40][41][42]. Multiple sequence alignments for ITS, ACT, CHS-1, GAPDH, and TUB2 were constructed using MAFFT v.7.11 on the web server (https://mafft.cbrc.jp/alignment/server, accessed on 12 February 2023) with the default settings [64]. BioEdit v.7.0.9.0 was used for adjusting the sequences [65] and TrimAl software was used to trim aligned sequences automatically using the gappyout command. Maximum parsimony (MP) analysis was done using PAUP XSEDE [66]. Maximum likelihood (ML) analysis was performed on XSEDE with the GTR + Gamma model and 1000 replications using RAxML-HPC2 The Bayesian posterior probabilities analysis (BYPP) was carried out using a Markov Chain Monte Carlo (MCMC) algorithm using MrBayes on XSEDE [67]. In order to choose the best-fit evolutionary models for each dataset, jModeltest 2.1.10 and the Akaike Information Criterion (AIC) were employed on the CIPRES platform. Four MCMC chains were run from random trees for 1,000,000 generations and sampled every 100th generation. The first 25% of the generated trees were ignored as burn-in and the remaining trees were used for analyzing the posterior probabilities. Gaps were considered missing data, and ambiguously aligned parts were eliminated. The phylogenetic trees were visualized in FigTree v.1.4.0 [68], and were further edited in Adobe Illustrator CC 22.0.0 (Adobe Systems, San Jose, CA, USA).  Type strains are indicated with "*". The isolates sequenced in this study are in bold.

Pathogenicity Assay
Koch's postulates were applied according to the procedures demonstrated by Bhunjun et al., (2021) to confirm the pathogenicity of our isolates [69]. Three replicates of detached leaves from an organic farm were considered for both wounded and non-wounded assays using mycelial plug incubation because of the lack of culture sporulation. The pineapple leaves were surface sterilized by washing them in 70% ethanol for 2 min, then in 2% sodium hypochlorite for 2 min, followed by three washes with sterile distilled water and laminar air drying. We chose a pineapple leaf instead of fruit to accurately assess symptoms and fungal spread through the host. Mycelial plugs were obtained from the fresh colonies grown on PDA (10-day colonies). Control inoculations were performed using uninoculated PDA plugs. In a moist chamber at 28 • C and 80% relative humidity, the inoculated and control leaves were incubated. Koch's postulates were confirmed by re-isolating the fungus from the infected leaves. The re-isolated fungus was identified based on cultural and morphological features.

Data Availability Statement:
Publicly available datasets were analyzed in this study. These data can be found here: https://www.ncbi.nlm.nih.gov (accessed on 12 November 2022).